Ginsenoside-Rg1 is one of the pharmacologically active components isolated from ginseng. It was reported that Rg1 protected dopamine (DA) neurons in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) models in vivo and in vitro. Our previous study also demonstrated that iron accumulation was involved in the toxicity of 6-OHDA. However, whether Rg1 could protect DA neurons against 6-OHDA toxicity by modulating iron accumulation and iron-induced oxidative stress is not clear. Therefore, the present study was carried out to elucidate this effect in 6-OHDA-treated MES23.5 cells and the possible mechanisms were also conducted. Findings showed Rg1 restored iron-induced decrease in mitochondrial transmembrane potential in MES23.5 cells, and increased ferrous iron influx was found in 6-OHDA-treated cells. Rg1 pretreatment could decrease this iron influx by inhibiting 6-OHDA-induced up-regulation of an iron importer protein divalent metal transporter 1 with iron responsive element (DMT1 + IRE). Furthermore, findings also showed that the effect of Rg1 on DMT1 + IRE expression was due to its inhibition of iron regulatory proteins (IRPs) by its antioxidant effect. These results suggested that the neuroprotective effect of Rg1 against iron toxicity in 6-OHDA-treated cells was to decrease the cellular iron accumulation and attenuate the improper up-regulation of DMT1 + IRE via IRE/IRP system. This provides new insight to understand the pharmacological effects of Rg1 on iron-induced degeneration of DA neurons.